KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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H2AX Directly Interacts with BRCA1 and BARD1 via its NLS and BRCT Domain Respectively in vitro

H2AX의 BRCA1 NLS domain과 BARD1 BRCT domain 각각과의 in vitro 상호 결합

  • Bae, Seung-Hee (Department of Microbial Engineering, Konkuk University) ;
  • Lee, Sun-Mi (Department of Microbial Engineering, Konkuk University) ;
  • Kim, Su-Mi (Functional Genoproteome Research Centre, Konkuk University) ;
  • Choe, Tae-Boo (Department of Microbial Engineering, Konkuk University) ;
  • Kim, Cha-Soon (Division of Radiation Effect Research, Radiation Health Research Institute, Korea Hydro & Nuclear Power Co., LTD.) ;
  • Seong, Ki-Moon (Division of Radiation Effect Research, Radiation Health Research Institute, Korea Hydro & Nuclear Power Co., LTD.) ;
  • Jin, Young-Woo (Division of Radiation Effect Research, Radiation Health Research Institute, Korea Hydro & Nuclear Power Co., LTD.) ;
  • An, Sung-Kwan (Department of Microbial Engineering, Konkuk University)
  • 배승희 (건국대학교 미생물공학과) ;
  • 이선미 (건국대학교 미생물공학과) ;
  • 김수미 (건국대학교 유전단백체 기능제어연구센터) ;
  • 최태부 (건국대학교 미생물공학과) ;
  • 김차순 ((주)한국수력원자력 방사선보건연구원) ;
  • 성기문 ((주)한국수력원자력 방사선보건연구원) ;
  • 진영우 ((주)한국수력원자력 방사선보건연구원) ;
  • 안성관 (건국대학교 미생물공학과)
  • Published : 2009.08.29
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Abstract

H2AX, a crucial component of chromatin, is implicated in DNA repair, cell cycle check point and tumor suppression. The aim of this study was to identify direct binding partners of H2AX to regulate cellular responses to above mechanisms. Literature reviews and bioinformatical tools were attempted intensively to find binding partners of H2AX, which resulted in identifying two potential proteins, breast cancer-1 (BRCA1) and BRCA1-associated RING domain 1 (BARD1). Although it has been reported in vivo that BRCA1 co-localizes with H2AX at the site of DNA damage, their biochemical mechanism for H2AX were however only known that the complex monoubiquitinates histone monomers, including unphosphorylated H2AX in vitro. Therefore, it is important to know whether the complex directly interacts with H2AX, and also which regions of these are specifically mediated for the interaction. Using in vitro GST pull-down assay, we present here that BRCA1 and BARD1 directly bind to H2AX. Moreover, through combinational approaches of domain analysis, fragment clonings and in vitro binding assay, we revealed molecular details of the BRCA1-H2AX and BARD1-H2AX complex. These data provide the potential evidence that each of the BRCA1 nuclear localization signal (NLS) and BARD1 BRCA1 C-terminal (BRCT) repeat domain is the novel mediator of H2AX recognition.

본 연구에서는 H2AX의 생리학적인 기능 및 분자세포 생물학적 기전 해석에 대한 보다 명확한 정보를 제시하고자, H2AX 관련 단백질들을 literature review 및 생물정보학적인 기술을 이용하여 최적의 결합 단백질체를 40개를 예측하곤 이들 가운데 상호작용 가능성이 높은 BRCA1와 BARD1 단백질을 선별하여 in vitro 결합실험을 통해 이를 증명하였다. 이들 두 가지의 유전자를 발굴하여, 클로닝하였다. 클로닝된 유전자를 이용하여 두 가지 단백질을 발현 및 정제하였으며, 단백질들의 자체적인 구조에 의한 결합능력을 판단하기 위해 in vitro binding assay법을 실시하였다. 단백질의 구조적 안정과 비특이적 결합을 억제하는 detergent만이 포함된 상태에서, 구조학적 및 물리학적 상호 결합의 유무를 판정할 수 있게 하였으며, BRCA1과 BARD1은 모두 H2AX에 결합함을 확인하였다. 이런 실험결과를 바탕으로 각각의 단백질에 대해 H2AX와의 최적 결합 부위를 알아내기 위해 각 유전자의 domain을 생물정보학적으로 분석하였다. 이에 RING domain, NES, NLS 및 BRCT domain에 해당하는 유전자 부분을 새로 클로닝하여, 다시 in vitro 결합실험 및 실험결과에 대한 literature review를 통한 분석을 실시한 결과, H2AX는 BRCA1의 NLS, BARD1의 BRCT domain 부분과 결합하는 것을 확인하였다. H2AX에 대한 BRCA1과 BARD1과의 결합은 DNA repair에 있어 BRCA1의 NLS와 BARD1의 BRCT domain을 통해 H2AX foci의 관련 세포 신호전달 기전에 중요한 역할을 하여 전체적으로 genomic stability에 영향을 미칠 가능성이 농후할 것으로 사료된다.

Keywords

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